1. Field of the Invention
The invention generally relates to the transport of low molecular weight enzyme substrates across lipid membranes of cells and, in particular, to the intracellular transport of fluorescent enzyme substrates.
2. Background of the Technology
It is a general goal of the industry to perform intracellular assays that measure enzyme activity such as enzyme expression and/or enzyme regulation. The enzyme activity can be a direct component of cellular pathways (e.g., hyperexpression of the enzyme due to a specific pathology), or a reporter of cellular function (e.g., reporter gene expression of the enzyme to measure a linked cellular event). In order to perform intracellular assays, improved enzyme-activated, noncytotoxic substrates that efficiently cross the cell membrane are desired. It is also desirable that the assay method enables greater detection sensitivity, a wide dynamic range of detection sensitivity, and ease of cell loading in a nondegradative method for adaptation to high throughput screening applications, as well as other biological and diagnostic assay formats. In view of the foregoing, there is a need for methods and compositions for facilitating the delivery of fluorescent enzyme substrates by complexing with a transporter to detect a variety of enzymes, such as β-glycosidases, esterases, proteases, phosphatases, and oxidases, into cells.
Many techniques have been developed to deliver small and macro molecules into living cells for monitoring or modifying intracellular processes. Most of these methods fall into one of three categories: direct transfer methods, carrier-mediated transfer, and transient cell membrane permeabilization transfer. See, for example, Stephens et al., “The Many Ways to Cross the Plasma Membrane”, Proc. Nat. Acad. Sci. USA, 98:8, 4295-4298 (2001). Each of these methods has advantages and disadvantages. The most widely used direct transfer method is glass capillary microinjection where glass micropipettes with a small tip (0.5 mm) are used to inject substances into adherent cells. Microinjection can give transfer efficiencies and cell survival rates up to 100%. Microinjection, however, is technically demanding and has low throughput (e.g., 100-200 cells per experiment) and is limited to adherent cell types. A second transfer method involves permeabilization of the cell membrane using detergents, toxins, UV laser light, or electrical pulses to punch holes in the cell membrane. Once the cell membrane has been permeabilized, substances can move across the cell membrane through the artificial pores by passive diffusion. This approach does not rely on highly skilled manual technique and is amenable to high throughput assays. The drawbacks to this permeabilization method, however, include <50% cell viability, irreversible permeabilization, and disrupted cellular functions of interest. For example, electroporation of cells requires cell suspension which disrupts cell cycle progression, cell adherence, and signal transduction processes.
A third type of transfer utilizes a cell-permeable molecule as a carrier to introduce substances (designated as the “cargo”) into cells. The carrier can be covalently coupled or fused to the cargo, or can complex with the cargo to carry the substance into the cell in a piggyback mode. Examples of attached or fused carriers include penetratin, VP22 protein (herpes simplex virus 1), and the TAT protein (HIV-1). A more general approach introduces large molecules (e.g., DNA, RNA or proteins) using cationic lipids such as DOTMA, DOTAP, and DOSPA. As with cell permeabilization methods, carrier-mediated transport is readily adaptable to high throughput assays with minimal technical skill requirements. However, transfer efficiencies often fall below 30% and can vary with cell type. In addition, liposomal delivery can significantly disrupt cellular functions such as lipid metabolism. None of the existing methods provides a complement of desirable transfer characteristics: simplicity, high transfer efficiency, high cell viability, general utility across cell types, and easy automation.
One of the most utilized carrier-mediated transfer methods is DNA transfection of cells using polycationic macromolecules. A variety of natural and synthetic polymers demonstrate an ability to complex with the polyanionic phosphate backbone of DNA to facilitate transport of the charge neutralized complex across the lipid bilayer. Examples of polycationic carrier molecules include spermadine, polylysine, polyethylenimine, “Starburst” PAMAM dendrimers, and polyvinylpyridinium salts such as PVPE and PVPEC. These carrier molecules have varying levels of cytotoxicity and transfection efficiencies. The dendrimers, in particular, exhibit lower cytotoxic effects with higher transfer efficiencies, and thus have been developed into commercially available DNA transfection agents (e.g., “SUPERFECT” and “POLYFECT” activated polyamine dendrimers, which are trademarks of Qiagen N.V.). These materials can serve as alternatives to liposomal carrier formulations. See, for example, Haensler et al., “Polyamidoamine Cascade Polymers Mediate Efficient Transfection of Cells in Culture” Bioconjugate Chem., 4, 372-379 (1993); Boussif et al., “A Versatile Vector for Gene and Oligonucleotide Transfer into Cells in Culture and in Vivo: Polyethyleneimine”, Proc. Natl. Acad. Sci. USA, 92, 7297-7301 (1995); Kabanov et al., “DNA Complexes with Polycations for the Delivery of Genetic Material into Cells” Bioconjugate Chem., 6, 7-20 (1995); Tang et al., “In Vitro Gene Delivery by Degraded Polyamidoamine Dendrimers”, Bioconjugate Chem., 7, 703-714 (1996); Kukowska-Latallo et al., “Efficient Transfer of Genetic Material into Mammalian Cells Using Starburst Polyamidoamine Dendrimers”, Proc. Natl. Acad. Sci. USA, 93:4897-4902 (1996).
Polycationic transport macromolecules have been used to transfer macromolecular substances, such as DNA plasmids, into cells. Several examples of transporting small molecular weight molecules having one to several anionic groups have appeared in the literature. Prestwich et al., for example, describe transporting membrane impermeable phosphatidylinositol polyphosphates and inositol polyphosphates into mammalian, plant, yeast, bacterial, and protozoal cells, using a polyamino dendrimer and type III-S histone as carriers. See Prestwich et al., “Intracellular Delivery of Phosphoinositides and Inositol Phosphates Using Polyamine Carriers”, Proc. Nat. Acad. Sci. USA, 97:11286-11291 (2000). The anionic lipids were fluorescently tagged for visualization by fluorescence microscopy. A similar approach has been described using amide and sulfonamide derivatives of the low molecular weight tris(aminoethyl)amine to carry anionic phospholipids as a complex into erythrocytes. Boon et al., “Facilitated Phosphatidylcholine Flip-Flop Across Erythrocyte Membranes Using Low Molecular Weight Synthetic Translocases”, J. Am. Chem. Soc., 123, 6221-6226 (2001). The transport decreases headgroup polarity of the phospholipids through complexation to promote diffusion of the phospholipids across the cell membrane. Tris(amidoethyl)amine- and tris(sulfonamidoethyl)amine-mediated transport of dilauroylphosphatidylcholine (DLPC) out of the cell reverts the echinocyte morphology (induced by cellular DLPC uptake) to the initial discocyte shape. The synthetic transporters also carried a fluorescent phosphatidylcholine probe (PC-NBD) into the cell.
U.S. Pat. Nos. 5,338,532; 5,527,524; and 5,714,166 disclose dendrimers (e.g., dense star polymers or starburst polymers) associated with a variety of materials. These materials include drugs, toxins, metal ions, radionuclides, signal generators, signal reflectors, chelated metal, signal absorbers, antibodies, hormones, biological response modifiers, diagnostic opacifiers, fluorescent moieties and scavenging agents. Processes for preparing the conjugates, compositions containing the conjugates and methods of using the conjugates are also disclosed in the aforementioned references.
Although various physical and biological methods have been used to introduce membrane-impermeable exogenous molecules into cells, none of the above-described methods disclose the transport of enzyme active fluorescent substrate complexes across a cell membrane. Moreover, none of the above-described methods disclose delivering a fluorescent substrate complex into a fixed cell for subsequent analysis of one or more fixed cellular components.
In view of the foregoing, there is a need for methods and compositions for facilitating the delivery of fluorescent substrates across the lipid membranes of cells.